Many reactions are characterized by the occurrence, or changes in level, of fluorescence when illuminated by a suitable excitation wavelength. In these types of reactions, a fluorescent sample absorbs light of a given wavelength and, in response thereto, emits light of a different wavelength.
Fluorescence may be inherent in the involved reagents or it may be provided deliberately by a suitable marker incorporated in the reactants. Hence, instruments for measuring fluorescence, fluorometers, are commonplace in the laboratory environment
Several difficulties exist with many stand alone fluorometers and those combined with other instrumentation. First, it is difficult to obtain very high intensity light in the proper wavelength from instruments which utilize a halogen or laser unitary light source without generating a large amount of heat. Similarly, since tungsten lights and the like must be on continuously to reach and operate under stable conditions, they also generate a large amount of heat. This large amount of heat can shorten the life of the lamp and should be dissipated because it may heat up the sample, thereby changing its fluorescent light emitting characteristics. Thus, these types of instruments require extensive cooling for the light source, and such light sources require frequent replacement.
In addition, for those instruments in which a group of samples may be tested simultaneously, a great deal of excitation light energy can be lost through diversion between the samples. This translates into a lower excitation efficiency.
Other fluorometers either lack sufficient sensitivity or are so expensive in construction as to be impractical for many purposes. For example, many fluorometers with a halogen light source do not have adequate sensitivity. This type of fluorometer has a limited dynamic range since all samples are illuminated and imaged at the same time if a typical charge coupled device (CCD) type camera is used. A laser light source type fluorometer can have better performance, but the laser light source is more expensive.
Many protocols, particularly in the broad field of microbiology, require repetitive, controlled temperature regimes. Apparatus filling this need are called “thermal cyclers”. It is useful to combine fluorometer with thermal cyclers for facilitating receipt of results which depend on fluorescence measurements to indicate reactions. One protocol which utilizes a thermal cycler is the polymerase chain reaction (PCR) (see U.S. Pat. No. 4,683,202 (Mullis)). To determine if amplification has occurred at the end of PCR, fluorescent dyes may be used as indicators, particularly intercalating dyes that fluoresce when bound to double stranded DNA but do not bind, or bind very inefficiently to single strands and have no or little signal in the presence of single strands.
Certain commercially available fluorometer/thermal cyclers can accommodate a standard ninety-six well tray of reaction tubes and include a fluorometer which uses a single, powerful incandescent light source to project light through an optical system to illuminate the tray and excite the fluorescent dyes therein to indicate positive reactions. Other commercially available instruments use a laser instead of an incandescent light source and a mechanical scanning device to isolate the signal from a reaction tube via a fiber optic cable onto a photodiode array.
FIG. 1 depicts a schematic of a system similar to certain commercially available instruments. It uses a single incandescent light source, lamp 1, which is a halogen projection lamp. Cooling is provided by a fan not shown. The light output passes through shutter 2 which is actuated by means not shown to shield the system when measurements are not being made. The light is directed toward the entirety of the sample tubes, not shown, held in sample plate 10 which is in intimate contact with a heating/cooling block 3 and in coordination with a data accumulation system not shown. Standard plates, or “trays”, used in this system hold 96 tubes. The directed beam of light passes through an excitation filter 4. The filtered light from filter 4 passes rectangular aperture 5 to confine the light to the sample tray area. Beamsplitter 6 reflects the light toward beam folding mirror 7. The light is then directed to fresnel lens 8. Fresnel lens 8 directs the light onto individual lenses mounted in plate 9, one lens per sample tube carried by plate 10. Once the light contacts the sample material any emitted light passes through beam splitter 6 and then through emission filter 11, lens 12, and into CCD type camera 13. CCD type camera 13 acquires an image of the entire sample tray. A computer program is used to calculate the fluorescent intensity of each sample tube from the image. The power measured by CCD type camera 13 indicates the reaction in the individual tube.
Other commercially available systems use an argon ion laser as the light source. In these systems, light from the argon ion laser passes through a dichroic mirror, a lens and a multiplexer which provides a fiber optic cable for each well of a 96-well plate. Excited light returns to the mirror and is reflected into a spectrograph which separates the light into a pattern that falls on a linear CCD detector. Appropriate filters are included in the optical paths.
Another commercially available system is partially depicted schematically in FIG. 1a. This instrument combines a “microvolume fluorometer” with thermal cycler 120. The light source in this instrument is a single light emitting diode (LED) 100 which projects light through lens and filter 102 to dichroic mirror 103 to reading lens 104 and then to the bottom of reaction tube 106. The reaction tubes, thirty-six in number, are held in carousel 108 which is stepped by motor 110 to place each tube in sequence over the reading lens 104. Any resultant emission reflects from mirror 103 and from one of the dichroic, color selective mirrors 114 through lenses 116 to one of photohybrids 118. A signal then is processed in an associated computer and printer not shown. The cycler 120 is air-cooled and heated using fan motor and associated fan blades 112 and heater coil 122 driving heated or cold air as directed by the program within the computer. Motor 124 positions the optical system as required. This system is not suited to a standard 8 by 12 tray and is expensive because it requires complex positioning mechanisms to present the tubes to the fluorometry system.
In view of the problems discussed above, there is a need to provide an inexpensive fluorometer having either a simple positioning mechanism or no positioning mechanism. In addition, there is a need for a fluorometer characterized by low heat-generating light sources using minimal power and which eliminates waste of excitation energy. There is also a need to provide light sources with rapid stabilization. Further, there exists a need for a highly sensitive fluorometer with an improved signal to noise ratio.